PB1712: LYOPHILIZATION BASED ISOLATION OF EXOSOMES FOR DELIVERY OF DRUGS AND PROTEIN
Authors: Fawad Ur Rehman, Rida e Maria Qazi, Sajid Zahra, Saba Naseem Awan, Syed Muhammad Areeb Ahmed, Afsar Mian
Journal: HemaSphere (2023)

Topic: 1. Acute lymphoblastic leukemia - Biology & Translational Research Background: Exosomes are extracellular vesicles having less than 200 nm size, secreted extracellularly by almost all types of cells. They regulate intercellular communication by delivering RNAi, proteins, viruses, and soluble factors as inherited payloads. In recent years, the application of exosomes to deliver small molecules have been widely investigated in tissue regeneration, therapeutics, as well as diagnostics. They are ideal candidates for cell engineering to deliver RNA interference (RNAi) molecules, plasmids, and CRISPR systems. However, the isolation of exosomes has remained challenging mainly due to limitations of specialized instruments and the least cost-effective reagents. In this study, we developed a novel method to isolate exosomes from cultured cells. Aims: The aim of this study was to develop a new method for the isolation of exosomes from cultured cells and testing them for drug and protein delivery and release in the cells. Methods: The supernatant from cultured cells was collected and subjected to lyophilization after low-speed centrifugation. The characterization and quantification of exosomes were performed using Bradford assay. Scanning Electron Microscopy (SEM) and Nanoparticles Tracking Analysis (NTA) were carried out for shape and size distribution. Western blot was performed for the evaluation of the exosome specific surface markers (CD63, CD9, CD81, calnexin, HSP70, and TSG101). The as-isolated exosomes were loaded with commercial dyes (Cy5, Eosin) for the evaluation of their drug delivery properties. Furthermore, their drug loading and release were confirmed by loading tyrosine kinase inhibitors (TKIs) as a payload. Finally, the anti-leukemic drugs (dasatinib and ponatinib) were loaded on the isolated exosomes for targeting leukemia cell line i.e., K562 and BCR-ABL expressing Ba/F3 cells in in vitro model, using MTT assay for cell proliferation and Annexin-V/PI for apoptosis detection. We also evaluated the effect of passive (incubation) and active (sonication, freeze-thaw, electroporation) cargo loading techniques for larger-size protein as payload. Results: Here we found that the isolated exosomes have round shape with an average size of ~130 nm. They expressed known exosome markers including CD63, CD9, CD81, calnexin, HSP70, and TSG101. These exosomes could efficiently target and deliver dyes (Eosin and Cy5) and TKIs (dasatinib and ponatinib) to K562 and Ba/F3-cells expressing BCR-ABL. The TKIs-loaded exosomes inhibited the growth of leukemic cells and induced apoptosis in a dose dependent manner. For loading and release of protein, the proteins were isolated by the immune-precipitation (IP) technique using paramagnetic Dyna beads. The isolated proteins were characterized by Bradford assay analysis. Electroporation was highly efficient among all cargo loading techniques, followed by sonication, whereas incubation was the least efficient method. The study has investigated the best modality for larger-size protein loading of exosomes as desired cargo for therapeutic effects in target tissues or cells that may serve as a potential model for Cas9 protein loading. treatment. Summary/Conclusion: We claim the development of a new method for active exosome isolation from cell culture. These exosomes could qualify for all the essential characterization, drug loading, and release ability by harnessing proliferation and inducing apoptosis in Ph+ leukemia cells. The study has also investigated the best modality for larger-size protein loading of exosomes as desired cargo for therapeutic effects in target tissues or cells that may serve as a potential model for Cas9 protein loading. treatment. Keywords: Leukemia, Nanoparticle